The Effect of Oral Iron Supplementation on Gut Microbial Composition: a Secondary Analysis of a Double-Blind, Randomized Controlled Trial among Cambodian Women of Reproductive Age

ABSTRACT The World Health Organization recommends untargeted iron supplementation for women of reproductive age (WRA) in countries where anemia prevalence is greater than 40%, such as Cambodia. Iron supplements, however, often have poor bioavailability, so the majority remains unabsorbed in the colon. The gut houses many iron-dependent bacterial enteropathogens; thus, providing iron to individuals may be more harmful than helpful. We examined the effects of two oral iron supplements with differing bioavailability on the gut microbiomes in Cambodian WRA. This study is a secondary analysis of a double-blind, randomized controlled trial of oral iron supplementation in Cambodian WRA. For 12 weeks, participants received ferrous sulfate, ferrous bisglycinate, or placebo. Participants provided stool samples at baseline and 12 weeks. A subset of stool samples (n = 172), representing the three groups, were randomly selected for gut microbial analysis by 16S rRNA gene sequencing and targeted real-time PCR (qPCR). At baseline, 1% of women had iron-deficiency anemia. The most abundant gut phyla were Bacteroidota (45.7%) and Firmicutes (42.1%). Iron supplementation did not alter gut microbial diversity. Ferrous bisglycinate increased the relative abundance of Enterobacteriaceae, and there was a trend towards an increase in the relative abundance of Escherichia-Shigella. qPCR detected an increase in the enteropathogenic Escherichia coli (EPEC) virulence gene, bfpA, in the group that received ferrous sulfate. Thus, iron supplementation did not affect overall gut bacterial diversity in predominantly iron-replete Cambodian WRA, however, evidence does suggest an increase in relative abundance within the broad family Enterobacteriaceae associated with ferrous bisglycinate use. IMPORTANCE To the best of our knowledge, this is the first published study to characterize the effects of oral iron supplementation on the gut microbiomes of Cambodian WRA. Our study found that iron supplementation with ferrous bisglycinate increases the relative abundance of Enterobacteriaceae, which is a family of bacteria that includes many Gram-negative enteric pathogens like Salmonella, Shigella, and Escherichia coli. Using qPCR for additional analysis, we were able to detect genes associated with enteropathogenic E. coli, a type of diarrheagenic E. coli known to be present around the world, including water systems in Cambodia. The current WHO guidelines recommend blanket (untargeted) iron supplementation for Cambodian WRA despite a lack of studies in this population examining iron’s effect on the gut microbiome. This study can facilitate future research that may inform evidence-based global practice and policy.

The manuscript entitled "The effect of oral iron supplementation on gut microbial composition: A secondary analysis of a doubleblind, randomized controlled trial among Cambodian women of reproductive age" by Finlayson-Trick & Nearing et al studied the effect of iron supplementation on gut microbiome in 172 Cambodian women. The study design and the choice of the participants seem pertinent as well as the choice of the multiple statistical analyses performed by the authors. Overall, the authors could show that iron supplementation did not significantly alter gut microbial diversity. Nevertheless, small changes in the abundance of some bacterial genera were measured in response to iron supplementation. The authors propose that iron supplementation could cause the increase in enteropathogenic bacteria and harm the population treated. This study is of importance as WHO recommends iron supplementation without knowing its repercussion(s) on the risk of infection. Major: 1/ The authors measured a significant increase in Enterobacteriaceae upon ferrous bisglycinate supplementation while detecting an increase in bfpA positivity mainly in the case of ferrous sulfate supplementation. Could the author discuss this descrepency? Also, could they be more specific about which of these two results seems to be the most compelling? Also, while the authors study two types of iron supplementations, could they conclude or at least comment on which supplementation seem the most effective? 2/ Would it be possible that the quantification of genes by PCR is dependent on the overall number of bacteria in the stool sample? How was normalized the detection of genes such as bfpA? Minor: -In Figure 4, the colors displayed do not correspond to the legend of the figure (Purple shows increase, not decrease and orange shows decrease not increase). A better quality of the image as well as a better color choice would help the reader decrypt the figure presented.
-L328: Change "whereas atypical EPEC strains are contain eae" to "whereas atypical EPEC strains contain eae" Reviewer #2 (Comments for the Author): The manuscript "The effect of oral iron supplementation on gut microbial composition: A secondary analysis of a double-blind, randomized controlled trial among Cambodian women of reproductive age" by Finlayson-Trick et al. investigated the impact of oral iron supplementations on gut microbiota. This is an important area of research, laying the groundwork for evidence-based guidance around the management of iron deficiency anemia. However, the manuscript provides limited insight and lacks of several key microbiota analysis such as compositional analysis at different taxanomic levels.
General comments: -16S rRNA V6-V8 regions were targeted for sequencing instead of the commonly used V4-V5 regions. Any justification for the choice taken? -Around 10 subjects were removed per study arm due to poor sequencing outcome. What could be the contributing factor? - Figure 4 & 5, please include statistical analysis.
-Please include compositional analysis of gut microbiota at different taxanomic levels.
-It would be interesting to investigate the correlation between gut microbiota and the occurrence of gastrointestinal symptoms. -(Line 301 -303): 'We did observe, however, some evidence to suggest that ferrous bisglycinate significantly increased the relative abundance of Enterobacteriaceae, a finding consistent with other studies in this field'. Are there any references related to ferrous bisglycinate? -The dosage of elemental iron differs greatly between the supplemental forms. How much of iron could end up in the lower gastrointestinal tract?
Specific comments: - Figure 3: Please simplify the legend for figure B, merging the representation for week and sample type.

Reviewer #3 (Comments for the Author):
This article is well written and supports the conclusions of the study. I appreciate that the authors published what is essentially negative results and made available all data and code. However, there are several changes that should be made to improve the clarity of the conclusions. Specifically: Lines 37-42: Remove all instances of "significantly". If something significantly increases, just say increases. If something doesn't significantly increase, then it is OK to say trending. "Significantly" is over-used in scientific literature and unnecessary. Line 44: Remove "appears to". Line 57: Instead of "we hope" use language like This study can facilitate... Line 98-99: Just say, "and the results have been contradictory". No need for "of those that have". For Fig. 2: This is a confusing figure. It would make more sense to me if the weeks would be shown alongside each other rather than having "Change in X" on the y-axis (and it should be "Change in X between weeks 0 and 12"). Stars should be explained in the legend. I'm not sure why you compared the time changes between trial arms if you didn't mention it in the text (or the Kruskal-Wallis test for that matter). Fig. 3: "Sample Type" in legend should be something like "Trial arm and Week" Fig. 4: This is a busy figure and I'm having a hard time understanding why you chose to plot like this. The legend says "Ferrous sulfate increased the relative abundance of Enterococcus and Weissella" but for Weissella it looks like 15 increased while 20 decreased. I'm assuming you mean the average abundance increased? Also, it looks like the colors are switched. The orange lines look like they are decreasing and vice versa for purple. And for Shigella, the green lines definitely have slopes so I don't understand why they would be considered constant. I also don't understand the inclusion of the tables unless you were doing some kind of chi-square or Fisher test.

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Dear Editor,
The manuscript "The effect of oral iron supplementation on gut microbial composition: A secondary analysis of a double-blind, randomized controlled trial among Cambodian women of reproductive age" by Finlayson-Trick et al. investigated the impact of oral iron supplementations on gut microbiota. This is an important area of research, laying the groundwork for evidence-based guidance around the management of iron deficiency anemia. However, the manuscript provides limited insight and lacks of several key microbiota analysis such as compositional analysis at different taxanomic levels.
General comments: -16S rRNA V6-V8 regions were targeted for sequencing instead of the commonly used V4-V5 regions. Any justification for the choice taken? -Around 10 subjects were removed per study arm due to poor sequencing outcome.
What could be the contributing factor? - Figure 4  Lines 37-42: Remove all instances of "significantly". If something significantly increases, just say increases. If something doesn't significantly increase, then it is OK to say trending. "Significantly" is over-used in scientific literature and unnecessary. Line 44: Remove "appears to". Line 57: Instead of "we hope" use language like This study can facilitate... Line 98-99: Just say, "and the results have been contradictory". No need for "of those that have". For Fig. 2: This is a confusing figure. It would make more sense to me if the weeks would be shown alongside each other rather than having "Change in X" on the y-axis (and it should be "Change in X between weeks 0 and 12"). Stars should be explained in the legend. I'm not sure why you compared the time changes between trial arms if you didn't mention it in the text (or the Kruskal-Wallis test for that matter). 1

Spectrum Reviewer Comments
We would first like to thank the reviewers and editors for their time and contributions toward our manuscript. The critical feedback we have received has been considered and substantially improved our manuscript. Below we have noted our comments for each point the reviewers have made and the actions we have taken to address them.

Reviewer #1
The manuscript entitled "The effect of oral iron supplementation on gut microbial composition: A secondary analysis of a double-blind, randomized controlled trial among Cambodian women of reproductive age" by Finlayson-Trick & Nearing et al. studied the effect of iron supplementation on gut microbiome in 172 Cambodian women. The study design and the choice of the participants seem pertinent as well as the choice of the multiple statistical analyses performed by the authors. Overall, the authors could show that iron supplementation did not significantly alter gut microbial diversity. Nevertheless, small changes in the abundance of some bacterial genera were measured in response to iron supplementation. The authors propose that iron supplementation could cause the increase in enteropathogenic bacteria and harm the population treated. This study is of importance as WHO recommends iron supplementation without knowing its repercussion(s) on the risk of infection.
Major: 1. The authors measured a significant increase in Enterobacteriaceae upon ferrous bisglycinate supplementation while detecting an increase in bfpA positivity mainly in the case of ferrous sulfate supplementation. Could the author discuss this discrepancy? Also, could they be more specific about which of these two results seems to be the most compelling? Also, while the authors study two types of iron supplementations, could they conclude or at least comment on which supplementation seem the most effective? These discrepant findings are interesting and we have several comments to address this: First, by visually inspecting the relationships in our differential abundance analysis, we note that the effect size was relatively small and that our result with Enterobacteriaceae was driven by a minority of samples. Ultimately, we were limited by a small sample size in this study. Secondly, it is critical to note that these discrepant findings represent two very different scopes of analysis, namely a bacterial family and a specific bacterial gene. Additionally, the techniques used to generate these results (e.g., 16S rRNA sequencing and qPCR) have their own inherent biases, which makes comparison of these two results very challenging. ; the proportion of women who reported any adverse side effects at 12 weeks did not differ by intervention group (chi-square, p=0.72). These 2 are the main trial findings reported in our primary manuscript which is currently under review at The Journal of Nutrition [revisions were submitted in early April]. The citation for the main trial manuscript will be updated in the text accordingly, upon acceptance for publication.

2.
Would it be possible that the quantification of genes by PCR is dependent on the overall number of bacteria in the stool sample? How was normalized the detection of genes such as bfpA?
The quantification of genes is dependent on the number of bacteria in the stool sample, so if there is more EPEC in the sample, there are more copies of the DNA detected. We extracted DNA from the same amount of stool between all the samples, which is standard practice and allows for fair comparison. We then normalized our samples against the positive DNA control and all the runs used the same volume and concentration of DNA for the positive sample.
Minor: 3. In Figure 4, the colors displayed do not correspond to the legend of the figure (Purple shows increase, not decrease and orange shows decrease not increase). A better quality of the image as well as a better color choice would help the reader decrypt the figure presented. We concur and have now included a high quality image with a revised legend (the colours now correspond with the legend).

Reviewer #2
The manuscript "The effect of oral iron supplementation on gut microbial composition: A secondary analysis of a double-blind, randomized controlled trial among Cambodian women of reproductive age" by Finlayson-Trick et al. investigated the impact of oral iron supplementations on gut microbiota. This is an important area of research, laying the groundwork for evidencebased guidance around the management of iron deficiency anemia. However, the manuscript provides limited insight and lacks several key microbiota analysis such as compositional analysis at different taxonomic levels. 2. Around 10 subjects were removed per study arm due to poor sequencing outcome. What could be the contributing factor? Due to natural variations in read count across samples, rarefaction is used to determine whether a specific sample has been sufficiently sequenced to represent its identity. This way all samples will have the sample number of reads, which allows for better comparison of diversity metrics. Based on our rarefaction curve (Fig. S2), we selected a sequencing depth of 5000 reads. Consequently, we removed approximately 10 subjects per study arm who did not meet this read cut off. A recent publication by Celis et al. (2022) entitled "Optimization of the 16S rRNA sequencing analysis pipeline for studying in vivo communities of gut commensals" provides further insight into this process. It is important to note that the same DNA extraction kit was used for all our samples, and we quantified our extracted DNA to ensure that approximately the same concentration was sent for sequencing.
3. Figure 4 & 5, please include statistical analysis. The data presented in Figure 4 and 5 are the statistically significant results generated through our differential abundance analysis. We have removed the tables to avoid confusion.
4. Please include compositional analysis of gut microbiota at different taxonomic levels.
In our current text we provide differential abundance data at the genera and family level ( Figure  4 and 5, respectively). We have added a stacked bar chart showing the relative abundance of each phyla within the samples organized by trial arm and week (Supplemental Figure 5).
5. It would be interesting to investigate the correlation between gut microbiota and the occurrence of gastrointestinal symptoms. We agree that this is an area of interest. In the manuscript reporting the main trial findings that is currently under review at The Journal of Nutrition, we report the adverse side effects reported among the participants. In summary, only 17% of women (73/441) reported any adverse side effects at 12 weeks (at the end of the trial); the proportion of women who reported any adverse side effects at 12 weeks did not differ by intervention group (chi-square, p=0.72). It was not the focus of the current manuscript, as we were limited by our smaller sample size for the 16S sequencing.
6. (Line 301 -303): 'We did observe, however, some evidence to suggest that ferrous bisglycinate significantly increased the relative abundance of Enterobacteriaceae, a finding consistent with other studies in this field'. Are there any references related to ferrous bisglycinate? After further consideration, we have removed this text "…a finding consistent with other studies in this field", as the research we were citing was conducted in mice. In their manuscript entitled "Iron supplements modulate colon microbiota composition and potentiate the protective effects of probiotics in dextran sodium sulfate-induced colitis," Constante et al. (2017) observed that in comparison to ferrous sulfate, mice that received ferrous bisglycinate (50mg/kg) had an increased abundance of members in the Bacteroidales order.
7. The dosage of elemental iron differs greatly between the supplemental forms. How much of iron could end up in the lower gastrointestinal tract? 7. Fig. 4: This is a busy figure and I'm having a hard time understanding why you chose to plot like this. The legend says "Ferrous sulfate increased the relative abundance of Enterococcus and Weissella" but for Weissella it looks like 15 increased while 20 decreased. I'm assuming you mean the average abundance increased? Also, it looks like the colors are switched. The orange lines look like they are decreasing and vice versa for purple. And for Shigella, the green lines definitely have slopes so I don't understand why they would be considered constant. I also don't understand the inclusion of the tables unless you were doing some kind of chi-square or Fisher test.
We have attempted to address the reviewers concerns with Figure 4 by removing the tables and correcting our legend. We have also better explained in the figure caption that the data presented is of the average relative abundance.
8. Fig. 5: Make the color scheme consistent with Fig. 4. The colour scheme in Figure 4 now matches the colour scheme in Figure 5.